We study the generation and regulation of respiratory rhythm in mammals. Breathing is a remarkable behavior in vertebrates that regulates gas exchange to support metabolism. A reliable and robust rhythm is essential for breathing in mammals. The failure to maintain a normal breathing rhythm in humans suffering from disorders such as sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, central alveolar hypo- ventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps sudden infant death syndrome, leads to serious adverse health consequences, even death. Various neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brain areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the site(s) and mechanisms for respiratory rhythmogenesis must be revealed. PreB"tzinger Complex (preB"tC) neurons in the brainstem underlie respiratory rhythm generation in vitro and are essential for breathing in awake adult rats in vivo. While our ultimate goal is to explain the generation of respiratory rhythm in intact mammals, in particular humans in health and disease, studies necessary to examine basic cellular/network mechanisms are presently impossible under in vivo conditions. In this proposal we will exploit a validated and powerful in vitro model of breathing, the rhythmic medullary slice generating a respiratory-related motor output, to investigate cellular properties of preB"tC neurons and advance our understanding of respiratory rhythmogenesis. We will use state of the art techniques such as calcium imaging and calcium uncaging to test the hypothesis: Calcium ion transients play a critical role in determining the dynamic properties of preB"tC neurons that are widely accepted to play a necessary role in respiratory rhythmogenesis. We propose 2 SPECIFIC AIMS. AIM 1: We will determine the temporal and somatodendritic distribution of calcium transients in preB"tC neurons. AIM 2: We will determine the effects of calcium transients on the dynamic properties of these key neurons. By detailing how intraneuronal calcium transients affect respiratory rhythm we will significantly improve our knowledge of neural control of breathing. These studies should make fundamental contributions to our understanding of breathing in humans in health and disease. PUBLIC HEALTH RELEVANCE: In humans, continuous breathing from birth is essential to life and requires that the nervous system generate a reliable and robust rhythm that drives inspiratory and expiratory muscles. The proposed studies will significantly advance our understanding of the neural mechanisms generating respiratory rhythm and shed light on human disorders of breathing.